Gene encoding bacterial beta-ketothiolase

ABSTRACT

The present invention is a method for controlling biopolymer synthesis by determining the genetics and enzymology of polyhydroxybutyrate (PHB) biosynthesis at the molecular level. The purified enzymes and genes provide the means for developing new PHB-like biopolymers having polyester backbones. Specific aims are to 1) control the chain length of the polymers produced in fermentation processes through genetic manipulation, 2) incorporate different monomers into the polymers to produce co-polymers with different physical properties, and 3) examine the physical/rheological properties of these new biopolymers in order to develop further design criteria at the molecular level. 
     The method for engineering biopolymer synthesis includes: isolation and characterization of the genes for the enzymes in the synthetic pathway (beta-ketothiolase, acetoacetyl-CoA reductase and PHB synthetase); cloning of the genes in a vector(s); placement of the vector(s) under the control of regulated promoters; expression of the genes; determination of the function and use of other factors such as substrate specificity in polymer production and composition; and isolation and physical and chemical analysis of the resulting polymers.

This invention was made with government support under Grant NumberNIH-5-R01GM33039 awarded by the National Institutes of Health andContract Number N0014-87K-0378 by the Navy. The government has certainrights in the invention.

This is a divisional of U.S. Ser. No. 08/297,667 entitled "Gene EncodingBacterial Acetoacetyl-Coa Reductase" filed on Aug. 29, 1994, now U.S.Pat. No. 5,512,669, by Oliver P. Peoples and Anthony J. Sinskey, whichis a continuation of U.S. Ser. No. 08/124,570 filed on Sep. 20, 1993,now abandoned, which is a continuation of U.S. Ser. No. 07/944,488 filedon Nov. 3, 1992, which is a divisional of U.S. Ser. No. 07/566,535 filedon Aug. 13, 1990, now U.S. Pat. No. 5,229,279, which is a continuationof U.S. Ser. No. 07/067,695 filed on Jun. 29, 1987, now abandoned.

BACKGROUND OF THE INVENTION

Synthesis by bacteria has long been the only means for production ofmany of the more complex biopolymers. Only recently have pathways forthe synthesis of these polymers been determined. Much effort has goneinto the isolation of the various enzymes and cofactors involved inthese pathways. Regulation of their expression has largely beenempirical, i.e., the concentration of nutrients or other factors such asoxygen level have been altered and the effect on polymer production andcomposition measured.

In order to have control over the production of these complexbiopolymers, and to modify them in a controlled fashion, it is necessaryto design a system for determining the chemical steps required for theirsynthesis; isolate and characterize the proteins responsible for thesechemical steps; isolate, sequence, and clone the genes encoding theseproteins; and identify, characterize, and utilize the mechanisms forregulation of the rate and level of the expression of these genes.

Polyhydroxybutyrate, a commercially useful complex biopolymer, is anintracellular reserve material produced by a large number of bacteria.Poly-beta-hydroxybutyrate (PHB), the polymeric ester ofD(-)-3-hydroxybutyrate, was first discovered in Bacillus megaterium in1925. Both the chemical and physical properties of this unique polyesterhave made it an attractive biomaterial for extensive study. PHB has avariety of potential applications, including utility as abiodegradable/thermoplastic material, as a source of chiral centers forthe organic synthesis of certain antibiotics, and as a matrix for drugdelivery and bone replacement. In vivo, the polymer is degradedinternally to hydroxybutyrate, a normal constituent of human blood.

PHB accumulates inside the cell as discrete granules stainable withSudan Black dye. The granules, which appear to be membrane bound,consist of approximately 98% PHB, 1-2% protein and approximately 0.5%lipid.

The enzymatic synthesis of the hydrophobic crystalline PHB granules fromthe C₂ biosynthon Acetyl-CoA has been studied in a number of bacteria.Three enzymes: beta ketothiolase, acetoacetyl-CoA reductase and PHBsynthetase, are involved in the conversion of Acetyl-CoA to PHB, asillustrated in FIG. 1.

Beta-Ketothiolase (acetyl-CoA-CoA-C-acetyl-transferase, E.C. 2.3.1.9)has been studied in A. beijerinckii (Senior and Dawes, Biochem. J., 134,225-238(1973)), A. eutrophus (Oeding and Schlegel, Biochem. J., 134,239-248 (1973)), Clostridium pasteurianum (Bernt and Schlegel, Arch.Microbiol., 103, 21-30(1975)), and Z. ramigera (Nishimura et al., Arch.Microbiol., 116, 21-27(1978)). However, the beta-ketothiolase enzyme hasnot been purified to homogeneity by any of these groups.

The best characterized Acetoacetyl-CoA reductase is that from Zoogloea,described by Saito et al., Arch. Microbiol., 114, 211-217(1977) andTomita et al., Biochemistry of Metabolic Processes, 353, D. Lennon etal., editors (Elsevier, Holland, 1983). This NADP-specific 92,000molecular weight enzyme has been purified by Fukui, et al., tohomogeneity, although only in small quantities.

PHB polymerase is not well characterized at present. When Griebel andMerrick, J. Bacteriol., 108, 782-789 (1971) separated the PHB polymerasefrom native PHB granules of B. megaterium there was a complete loss ofenzyme activity. They were able to reconstitute activity only by addingPHB granules to one of two fractions of the protein. More recently,Fukui et al., Arch. Microbiol., 110, 149-156(1976) and Tomita et al.(1983), investigated this enzyme in Z. ramigera and partially purifiedthe non-granule bound PHB polymerase.

Despite the diversity of the producing organisms, the composition andstructure of the PHB polymer remain constant. In contrast, the molecularweight is reported to vary from species to species, ranging from50,000to 1,000,000 Daltons. The intrinsic or extrinsic mechanisms thatdetermine this aspect of the polymer synthesis are still unclear.

PHB biosynthesis is promoted under a variety of nutrient limitingconditions. For example, Azotobacter beijerinckii, a nitrogen fixingbacteria accumulates up to 70% dry cell weight as PHB when grown onglucose/ammonium salts under limiting oxygen. Increasing the availableoxygen leads to a decrease in PHB synthesis and a concomittant reductionin the levels of two of the biosynthetic enzymes. The reduction inenzyme levels is indicative of a regulatory mechanism(s) operating atthe genetic level. Nitrogen limitation of the growth of Alcaligeneseutrophus results in yields of up to 80% dry cell weight PHB. Similarly,Halobacterium and Pseudomonas sp. increase PHB production under nitrogenlimitation. Determining the mechanisms by which PHB synthesis isstimulated could lead to novel control strategies for synthesis of PHB.

Given the extremely high yields of this polymer obtainable throughclassic fermentation techniques, and the fact that PHB of molecularweight greater than 10,000 is useful for multiple application, it isdesirable to develop new PHB-like biopolymers to improve or create newapplications. Different PHB-like polymers with altered physicalproperties are occasionally synthesized by bacteria in nature. Ingeneral, the bacteria incorporate monomers other thanD(-)hydroxybutyrate into the final polymer product. These alternatesubstrates are presumably incorporated through the enzymes of the normalPHB biosynthetic pathway. Unfortunately, it is difficult to study thebiosynthesis of these polymers since they are produced underuncontrolled conditions by an indeterminate number of bacterial species.

The production of poly-beta-hydroxyalkanoates, other than PHB, bymonocultures of A. eutrophus and Pseudomonas oleovorans has recentlybeen reported by deSmet, et al., in J. Bacteriol., 154, 870-878(1983)and Senior, et al., Eur. Patent Appl. 86303558.0. In both bacteria, thepolymers were produced by controlled fermentation. A. eutrophus, whengrown on glucose and propionate, produces a heteropolymer of PHB-PHVpoly B-hydroxyocleic acid, PHV content reaching approximately 30%. P.oleovorans produces a homopolymer of poly-beta-hydroxyoctanoate whengrown on octane. Nocardia has been reported to form copolymers ofPHB-PH-2-butenoate when grown on n-butane. Determination of the finalcomposition of 3-hydroxybutyrate polymers by controlled fermentationusing selected substrates is also disclosed in U.S. Pat. No. 4,477,654to Holmes et al.

Despite the great interest in synthesis of biopolymers and especiallyPHB, the mechanism and genetics of how the biosynthesis ofheteropolymers occurs is unknown. To date, the only genetic studies onPHB synthesis have been limited to isolation of PHB-mutants of A.eutrophus by Schlegel et al., Arch. Microbiol. 71, 283-294 (1970).

It is therefore an object of the present invention to provide a methodfor determining the chemical steps required for synthesis of complexbiopolymers, particularly PHB and PHB-like polymers, for isolating andcharacterizing the proteins responsible for these chemical steps, forisolating, sequencing, and cloning the genes encoding these proteins,and for identifying, characterizing, and utilizing the mechanisms forregulation of the rate and level of the expression of these genes.

It is another object of the present invention to provide purifiedproteins expressed from the genes encoding the proteins for synthesis ofpoly-hydroxybutyrate.

It is still another object of the present invention to provide sequencesregulating the expression of the genes encoding the proteins requiredfor biopolymer synthesis.

It is a further object of the present invention to provide methods forusing these proteins and regulatory sequences to create novelbiopolymers having polyester backbones.

SUMMARY OF THE INVENTION

The present invention is a method for controlling biopolymer synthesisby determining the genetics and enzymology of polyhydroxybutyrate (PHB)biosynthesis at the molecular level. The purified enzymes and genesprovide the means for developing new polyester PHB-like biopolymers.Specific aims are to 1) control the chain length of the polymersproduced in fermentation processes through genetic manipulation, 2)incorporate different monomers into the polymers to produce co-polymerswith different physical properties, and 3) examine thephysical/rheological properties of these new biopolymers in order todevelop further design criteria at the molecular level.

The method for engineering biopolymer synthesis includes: isolation andcharacterization of the genes for the enzymes in the synthetic pathway(beta-ketothiolase, acetoacetyl-CoA reductase and PHB polymerase);cloning of the genes in a vector(s); placement of the vector(s) underthe control of regulated promoters; expression of the genes;determination of the function and use of other factors such as substratespecificity in polymer production and composition; and isolation andphysical and chemical analysis of the resulting polymers.

Genes from Gram negative organisms, Zoogloea ramigera strain I-16-M,Alcaligenes eutrophus and Nocardia salmonicolur, were identified orisolated and used to study the PHB biosynthetic pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the PHB biosynthetic pathway.

FIG. 2 is a restriction map of pUCDBK1 and diagram of the constructionof pZT3.5.2 and pZR14.

FIGS. 3a and 3b is the Zoogloea thiolase gene sequence. The sequenceslocated at positions hemologeous to the E. coli "-10"0 and "-35"concensus regional (-100 to -95 and -122 and -116) upstream from thetranscription start site (bold arrow) are underlined. A probableribosome binding site is underlined (-11 to -8).

FIG. 4 is a diagram of the extent of the 5' deletions in plasmidconstructs pZT3-pZT3.5.2 and thiolase specific activity (units/mgprotein) expressed following induction of tac-directed expression.

FIG. 5 is a diagram of the construction of reductase expression plasmidspZT1, pZT2 and pZT3.

FIG. 6a and 6b are is the complete nucleotide sequence of the a2.5 Kb ofZ. ramigera DNA located downstream from the thiolase gene in clonepUCDDK1. The sequence of 2094bp extending from the first Sal 1 site tothe second Sma 1 site is shown. Also shown is the translation product ofthe acetoacetyl-CoA reductase structural gene extending from the ATG atnucleotide 37 to the TGA stop codon at nucleotide 760. Amino acidresidues 2 to 6 are underlined. These amino acids are identical to thoseobtained by Edman degradation of the purified protein. Restriction sitesfor Sal 1 and Sma 1 are shown.

FIG. 7 is a diagram of the construction of overproduction vectors pATand pAR.

FIGS. 8 a, 8b, 8c, and 8d shows the nucleotide sequence of the 2 Kbfragment A. eutrophus DNA cloned in plasmid pAeT3. The translationproducts of the A. eutrophus thiolase and acetoacetyl-CoA reductasegenes extending from nucleotides 40 to 1219 and 1296 to 2034,respectively, are shown. Restriction endonuclease cleavage sites used inthe construction of the over production vectors pAT and pAR are shown.Pst 1=Pst 1; Ava=2 and Dde=Dde 1.

FIG. 9 are representative acyl thiolester substrates for PHB polymeraseand the proposed repeating units in the resulting monomer.

DETAILED DESCRIPTION OF THE INVENTION

Poly(beta-hydroxybutyrate) (PHB) is a unique biodegradable thermoplasticproduced in a fermentation process. PHB has a number of interestingproperties which make it an attractive material for the plastic andbiomedical industries. One feature of PHB is that alternate polymers canbe produced by carefully controlling the fermentation conditions. This,in addition to the well characterized biochemistry of the biosyntheticpathway, makes it a particularly useful system for demonstrating amethod of biopolymer engineering using recombinant biotechnology.

There are three enzymes in Z. ramigera responsible for PHB synthesis: athiolase, a reductase, and a polymerase. Thiolases are ubiquitousenzymes which catalyze the synthesis and cleavage of carbon-carbon bondsand thus occupy a central role in cellular metabolism. Differentthiolase enzymes are involved in terpenoid, steroid, macrolide and otherbiosynthetic pathways as well as the degradation of fatty acids. Theonly thiolase genes cloned to date (from rat mitochondria and E. coli)have been for thiolases involved in fatty acid degradation which areregulated at the level of gene expression. In Z. ramigera, thecondensation of two acetyl-CoA groups to form acetoacetyl-CoA iscatalyzed by beta-ketothiolase. The Acetoacetyl-CoA is then reduced byan NADP-specific reductase to form D(-)-beta-hydroxybutyryl-CoA, thesubstrate for PHB polymerase. The reductase involved in PHB biosynthesisin Z. ramigera is stereospecific for the D(-)-isomer ofhydroxybutyryl-CoA and uses NADP(H) exclusively as a cofactor. The PHBpolymerase in Z. ramigera is stereospecific for D-beta-hydroxybutyrylCoA. Polymerase from other bacteria such as A. eutrophus should utilizeother substrates, for example, D-beta-hydroxyvaleryl CoA since additionof propionate into A. eutrophus cultures leads to incorporation of C₅and C₄ units into a PHB/HV copolymer.

By combining these enzymes with the appropriate substrates undercontrolled culture conditions of available oxygen and temperature, avariety of polymers can be constructed. The enzymes or nucleotidesequences controlling their expression can also be modified to alter thequantity of expression or substrate specificity to further vary theresulting polymers. An added advantage to the present invention is thatsubstrates which normally cannot be used with whole cells can bemanufactured using the isolated enzymes.

The following methods are used to isolate genes encoding beta-ketothiolase, AcetoAcetyl-CoA reductase, and PHB polymerase and theirexpression products, to identify and characterize sequences regulatingtheir expression, and to determine the effect of culture conditions andsubstrate availability on polymer production. Techniques forconstructing systems for the production of PHB and PHB-like biopolymersare also disclosed.

Construction of a Z. ramigera Library

Zoogloea ramigera strain I-16-M was used initially to study the geneticsof the PHB biosynthetic pathway. Z. ramigera DNA was purified from 200ml mid-log phase cultures as follows: cells were harvested bycentrifugation, washed in 20 mM Tris-HCl, pH 8.2, and resuspended in 10ml of Tris-HCl. The cells were then spheroplasted by the addition of 10ml of 24% w/v polyethylene glycol 8000 and 2 ml of 25 mg/ml lysozyme,followed by incubation at 37° C. for 30 min. The spheroplasts wereharvested by centrifugation, resuspended in 5 ml of TE buffer (10mMTris-HCl, pH 8.0, 1 mM EDTA), 300 microliters of 10% w/v SDS added,and the cells lysed by incubating at 55° C. for 10 min. An additional 10ml of TE was added and the lysate incubated with RNAse (50 microgram/ml)and proteinase K (30 microgram/ml) for 1 h at 37° C. The DNA was thenpurified by CsC1 gradient centrifugation.

Plasmid DNA preprations were carried out using the method of Birnboimand Doly in Nucleic Acids Res., 7, 1513-1523(1979) as described byIsh-Horowicz and Burke, Nucleic Acids Res., 9, 2989-2998(1981). LambdaDNA was prepared by standard procedures described in Maniatis et al.,Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. 1982).

A recombinant library of random Z. ramigera DNA fragments wasconstructed using the lambda gt11 expression vector described by Youngand Davis, Science, 222, 778-782(1983). Z. ramigera DNA was firstmethylated using EcoRI methylase and then partially digested with DNAse1in the presence of Mn²⁺, as taught by Anderson, Nucleic Acids, 9,3015-3026(1981). The ends of the partially digested DNA fragments wererepaired using Klenow polymerase, EcoRI linkers added, and the DNAdigested to completion with an excess of EcoRI. Fragments of 2-8 kb weresize-selected on a 1.5% agarose gel, purified by electroelution, andligated with EcoRI-digested, phosphatased lambda gt11 DNA. Ligationswere carried out for 18 h at 4° C. using 2 micrograms of lambda gt11 DNAand 1 microgram of target DNA in a total volume of 10 microliters. Thecomplete ligation reactions were packaged in vitro using lambda extractsprepared from E. coli strains BHB2688 and BHB2690, Hohn and Murray,Proc. Natl. Acad. Sci. USA, 74, 3259-3263(1977), as described byManiatis et al. (1982). Packaged phage were plated out and amplified onE. coli Y1088.

Screening of the lambda gt11 expression library was carried out usingrabbit anti-thiolase anti-bodies and a modification of the proceduredescribed by Young and Davis, Science, 222, 778-782(1983).

Identification of the Z. ramigera Thiolase Gene

Thiolase antiserum was prepared in New Zealand White female rabbits,using purified thiolase protein by standard procedures. Antibody titerwas estimated by the Ouchterlony double-diffusion assay, Acta Pathol.Microbiol. Scand., 26, 507-515(1949). Purified antibody was preparedfrom the serum by chromatography on protein A agarose according toBighee et al., Mol. Immunol., 20, 1353-1357(1983). Approximately 4×10⁴recombinant phage adsorbed to E. coli Y1090 were plated out on 15 cmLB-agar plates and incubated at 42° C. for 3 h. The plates were thenoverlayed with nitrocellulose filters (Schleicher & Schull, BA85), whichhad previously been saturated in 10 mM IPTG, and incubated a further 4 hat 37° C. Filters were removed, washed for 10 min in TBST (50 mMTris-HCl, pH 7.9, 150 mM NaCl, 0.05% Tween-20), incubated in TBST plus20% v/v fetal calf serum for 30 min, and rinsed in TBST. First antibodywas bound by incubating the filters in 10 ml TBST plus purifiedanti-thiolase antibody (10 microliters) for 1 h at room temperature. Thefilters were subsequently washed in three changes of TBST for 5 min eachtime. Bound first antibody was detected using a biotin-avidinhorseradish peroxidase detection system (Clontech Laboratories) andhorseradish peroxidase color development reagent (Bio-Rad Laboratories,Richmond, Va.).

Proteins were separated by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis according to the method of Laemmli, Nature, 222,680-685(1970) and electrophoretically transferred to nitrocellulosefilters (Schleicher & Schuill BA85), essentially as described byBurnette, Anal. Biochem., 112, 195-203(1981). Following transferovernight at 30 V, filters were rinsed in TBS (TBST without Tween-20)and incubated in TBS plus 5% bovine serum albumin. Proteins reactingwith anti-thiolase serum were then detected by incubating the filters in100 ml of TBS, 1% gelatin containing 2 ml of anti-thiolase serum for 1-2h. Bound first antibody was subsequently detected using goat anti-rabbitIgG horseradish peroxidase conjugate and horseradish peroxidase colordevelopment reagent (Bio-Rad Laboratories, Richmond, Calif.).

DNA blots were prepared using DNA fragments separated on agarose gels bythe sandwich blot method of Smith and Summers, Anal. Biochem., 109,123-129(1980) based on the technique developed by Southern, J. Mol.Biol., 98, 503-517(1975). Filters were hybridized with DNA probeslabeled to a high specific activity (0.1-1×10⁸ cpm/microgram of DNA)with [alpha-³² P]dATP, by nick translation, Rigby et al., J. Mol. Biol.,113, 237-251(1977). Prehybridizations and hybridizations were carriedout at 65° C. in sealed polythene bags. Theprehybridization/hybridization solution contained 5×SSCP (1×SSCPcontains 0.15M NaCl, 0.15 M sodium citrate, 10 mM Na₂ HPO₄, 10 mM NaH₂PO₄), 5×Denhardt's solution, 0.1% (w/v) SDS, 10 mM EDTA, and 100microgram/ml sonicated denatured salmon DNA. Filters were prehybridizedfor 8-18 h and hybridized for 16-18 h using 10⁷ cpm of labeled DNA probeper filter.

Lysogens of lambda gt11 recombinant clones were prepared in E. coliY1089 as described by Young and Davis, Science 222, 778-782 (1983). Forthe preparation and analysis of lambda-coded proteins, lysogens weregrown at 30° C. in LB (100 ml) until they reached an OD₆₀₀ of 0.5. Theprophage was induced by a 20 min incubation at 45° C., IPTG added to 5mM and the induced lysogens incubated at 37° C. for 1 h. Cells wereharvested, resuspended in assay buffer (0.1M Tris-HCl, pH 7.5, 5 mMbeta-mercaptoethanol, 5% (v/v) glycerol), lysed by sonication, celldebris pelleted by centrifugation, and the cell extracts stored at -20°C. The protein concentrations of bacterial lysates were assayed by themethod of M. M. Bradford in Anal. Biochem. 72, 248-254 (1976), usingbovine serum albumin as a standard. Thiolase-enzyme assays wereperformed as described by Nishimura et al., Arch. Microbiol. 116, 21-27(1978).

DNA fragments were cloned into the M13 vectors mp10 and mpll andsequenced by the dideoxy chain-termination method of Sanger et al.,Nucleic Acids Res. 10, 141-158 (1980), Proc. Natl. Acad. Sci. USA 74,5463-5467 (1977). The M13 sequencing primer and other reagents werepurchased from Amersham Corp. G/C rich regions were resequenced usingdtTP in place of dGTP as described by Mills and Kramer, Proc. Natl.Acad. Sci. USA 76, 2232-2235 (1979). Computer-assisted sequence analysiswas accomplished using the Staden programs. (Nucleic Acids Res. 10,141-158 (1984).

Approximately 2×10⁵ recombinants were obtained from 1 microgram ofpurified target DNA, and amplified in E. coli Y1088. A total of 10⁵amplified phage were screened using purified rabbit anti-thiolaseantibodies. The initial screening identified 10 potentially positiveclones (LDBK1-LDBK10). Analysis by restriction digestions demonstratedthat clones LDBK2-10 are identical. Clones LDBK1 and LDBK2 were selectedfor further study. LDBK1 has an insert composed of 2 EcoRI fragments of3.6 kb and 0.75 kb. LDBK2 has an insert composed of 3 EcoRI fragments of1.65 kb and 1.08 kb.

The proteins coded for by the LDBK1 and LDBK2 insert sequences wereanalyzed both for thiolase-enzyme activity and for cross-reaction torabbit anti-thiolase serum. Lysogenic strains of E. coli Y1089containing LDBK1 and LDBK2 phage DNA were prepared. Several lysogenswere obtained for each clone and two of these, Y1089/LDBK1 andY1089/LDBK2, were used for subsequent studies. A lysogen of the lambdagt11 vector, BNN97/lambda gt11, was used as a control. The results ofthe thiolase-enzyme assays clearly indicate that the proteins fromY1089/LDBK1 contain a substantial amount of thiolase activity.Furthermore, the thiolase activity is inducible, greater than 5-fold, bythe addition of IPTG. This shows that expression of the thiolase-codingsequences is under the transcriptional control of the lac promotercontained in the lambda gt11 vector. Neither the Y1089/LDBK2 nor theBNN97/lambda gt11 protein lysates demonstrate any significantthiolase-enzyme activity even when induced with IPTG.

The size of the proteins responsible for the initial positive reactionto rabbit anti-thiolase antibodies was investigated by Western blotexperiments. Protein lysates were separated by SDS-polyacrylamide gelelectrophoresis, transferred to nitrocellulose filters, and screenedwith rabbit anti-thiolase serum. The results show an immuno-reactive40,000 dalton protein in both the IPTG-induced and non-IPTG-inducedlysate of Y1089/LDBK1.

The LDBK1 insert was restriction mapped. The large 3.6 kb EcoRIfragment, containing the complete thiolase gene region, was subclonedinto the plasmid vector pUC8 for ease of manipulation. Restrictionanalysis of one of the subclones obtained, pUCDBK1, shown in FIG. 2,confirmed the restriction map of this fragment in LDBK1. pUCDBK1 DNA waslabeled to a high specific activity with ³² p and hybridized tonitrocellulose filters containing Z. ramigera chromosomal DNA digestedwith the same enzymes used to restriction map pUCDBK1. Southernhybridization experiments confirm that the 5.4 kb genomic fragmenthybridizes to both a 1.45 kb SalI/EcoRI and 1.05 kb SalI fragment frompUCDBK1. Based on the result of Southern hybridization experiment, thecloned pUCDBK1 insert is represented in the Z. ramigera genome onlyonce.

DNA sequence analysis of the pUCDBK1 insert was carried out using theM13/Sanger dideoxy chain termination method. To locate the gene-codingregion, individual DNA sequences were scanned in all six reading framesfor homology to the NH₂ -terminal amino acid sequence. By using thisapproach, the gene-coding region within the 1.46 kb EcoRI-Sal/I fragmentwas identified. The complete nucleotide sequence of the plus strand ofthe gene is shown in FIGS. 3a and 3b 290 downstream from the EcoRI sitelies the start of the thiolase structural gene, assigned by comparingthe DNA sequence to the NH₂ -terminal amino acid sequence. The NH₂-terminal sequence lies in the single long open reading frame whichextends from position -89 to the stop codon (TAG) at nucleotide 1174.Beginning with a serine and extending for 25 residues, theexperimentally determined NH₂ -terminal sequence aligns identically withresidues 2 through 26 of the translated DNA sequence. Translation of theDNA sequence was then used to deduce the remaining amino acid sequencefrom residue 27 to 391 (nucleotides 79 to 1173). Hence, translation ofthe DNA sequence from nucleotide 1 to 1174 (in this reading frame)encodes a 391-amino acid polypeptide with a calculated M_(r) 40,598.This value is in very good agreement with that of M_(r) =42,000determined by SDS-polyacrylamide gel electrophoresis.

Two additional pieces of evidence confirm that this translation produceis the correct amino acid sequence of thiolase. First, a search of thepredicted amino acid sequence for the active site peptide(NH2--Gly--Met--Asn--Gln--Leu--Cys--Gly--Ser--Gly--COOH) located thispeptide at residues 84-92. Finally, the predicted amino acid compositionfrom the translation product and that determined experimentally are inexcellent agreement. The G/C content of the 1.46 kb EcoRI-SalI fragmentis high, 66.2%. When considered separately, the 5'-flanking 290 bp has aG/C content of 57.4% and the structural gene region 68.4%. The aminoacid sequence confirms that the Z. ramigera thiolase contains 5 cysteineresidues. The Z. ramigera active site cysteine is at residue Cys-89.Additional cysteines which may be involved in inter- or intradisulphidebonds are Cys-125, Cys-323, Cys-377, and Cys-387. NH₂ -terminal sequenceanalysis indicated a serine at position 1.

Seven nucleotides upstream from the ATG start codon is a potentialribosome-binding site, 5'-CTGAGGA-3' identified by homology to the E.coli sequence. Additional start codons including two GTGs which caninitiate translation in some bacterial genes are located furtherupstream. Examination of the 5'-flanking region for homology to the"-10" and "-35" E. coli promoter elements, identified a potential "-35'region at residues -122 to -116 and a corresponding "-10 region",5'-TATAAT-3', at position -100 to -95. A poly(T) tract at position -255to 31 266 is also present. It is clear that the only post-translationalprocessing of thiolase is the removal of the N-formylmethionine residue,as alternate start codohs, ATC or GTG, are either out of frame or havean in-frame stop codon before the start of the structural gene.

The 1.5 Kb Sal1-EcoR1 fragment from puCDBK1 contains the entire Z.ramigera thiolase structural gene plus 283 bp of 5'/flanking DNA. Aseries of plasmid constructions were made in which this DNA fragment wasinserted into the tac promoter vector pKK223-3 (or derivatives thereof).pZT3 was made by cleaving pUCKBK1 with Sal1, blunt-ending with Klenowpolymerase and adding on EcoR1 linkers. Following digestion with EcoR1,the 1.5 Kb fragment was purified from an agarose gel and inserted intothe EcoR1 site of pKK223-3. Recombinant clones having the gene insertedin the correct orientation with respect to the tac promoter wereidentified by restriction analysis following transformation of E. coliJM105.

A series of clones deleted of sequences in the 283 bp flanking the 5'end of the thiolase gene was then constructed. pUCDBK1 DNA was digestedwith EcoR1 and treated with Bal31 nuclease. Following Sal1 digestion,the ends of the fragments were repaired with Klenow and EcoR1 linkersadded on. The plasmid DNA was cleaved with EcoR1 and fragments in thecorrect size range, 1.2-1.4 Kb, purified from an agarose gel and ligatedinto the EcoR1 site of pKK223-3. Clones of interest were identified byrestriction mapping and the extent of the 5'-deletion determined by DNAsequencing, diagrammed in FIG. 4. From this series of clones,pZT3.1-pZT3.5, the clone with the largest deletion, pZT3.5, had 84 bp ofthe 5'-flanking DNA remaining and therefore a subsequent Bal31 deletionexperiment was carried out as follows: pZT3.5 DNA was digested tocompletion with EcoR1 and treated with Bal31 nuclease; the ends wererepaired using Klenow polymerase and EcoRI linkers ligated on, followingdigestion to completion with EcoR1 and BamH1, fragments corresponding tothe NH₂ -terminal region of thiolase were eluted from an agarose gel,ligated with BamH1-EcoR1 digested M13 mp 11 DNA and plated out on E.coli JM101; single-stranded DNA was prepared from 50 transformants andthe extent of the 5'-deletion analyzed by T-tracking; double-strandedDNA was prepared, in vitro, from clones of interest and the EcoR1 -Bam1inserts recovered by restriction digestion and elution from an agarosegel.

In order to reconstruct the intact thiolase gene, the 290 bpBamH1-HindIII fragment from pZT3.5 was ligated into a vector (pKK226)derived from pKK223-3 by deleting the BamH1 site upstream from the tacpromoter; this C-terminal vector was subsequently used for thereconstruction of the Bal31 deleted NH₂ -termini of interest; clonespZT3.5.1 and pZT3.5.2 were used in subsequent studies.

The effect of deleting sequences in the 283 bp of DNA flanking thethiolase ATG translation initiation codon was determined by analyzingthe level of thiolase activity produced by plasmids pZT3.1-pZT3.5.2. 100ml cultures of the E. coli JM105 containing each plasmid were inducedwith IPTG for 15 hours and the level of thiolase assayed. FIG. 4presents the results of this series of experiments and illustrates theextent of the 5'-deletions for each plasmid. The most notable feature ofthese results is the very high level of thiolase expression obtainedwith clones, pZT3.3-pZT3.5.2, the maximum obtained being 178 u/mg forpZT3.5. This represents an increase of 5.9-fold as compared to plasmidpZT3 which contains the entire 283 bp of 5'-flanking DNA. The datapresented in FIG. 4 demonstrate that the thiolase 5'-flanking sequenceslocated between -84 (pZT3.5) and -168 (pZT3.2) strongly inhibit theexpression of the thiolase gene from the tac promoter. Furthermore, thelocation of these sequences can be narrowed down to the region between-84 (pZT3.5) and -124 (pZT3.4) as the deletion of this region results inthe maximum level of tac-directed thiolase expression. Further deletionsto -37 (pZT3.5.1) and -26 (pZT3.5.2) do not increase the level ofthiolase expression, and in fact a slight decrease is observed. It isimportant to note that the time course of induction for this series ofclones follows the same kinetics as pZT3 and is not appreciably affectedby the deletions.

In order to determine if the thiolase promoter lies in the region-84(pZT3.5) to -124 (pZT3.4), S1 nuclease protection experiments werecarried out according to the method of Berk and Sharp, Cell 12, 721-732(1977) on Z. ramigera RNA. The RNA was isolated from a 100 ml mid-logphase culture by the hot phenol/glass beads extraction procedure ofHinnenbusch et al., J. Biol. Chem. 258, 5238-5247 (1983). 5'-³²P-labelled DNA probe was prepared as follows: 10 micrograms, plasmidpZT3.1 DNA was digested to completion with Aval and subsequently treatedwith CIP; the Aval restriction fragments were labelled at the 5'-endwith [gamma-³² P]-ATP and polynucleotide kinase; following EcoR1digestion, the DNA was separated on an 8% acrylamide gel and the ³²P-labelled 280 bp probe fragment eluted and recovered by ethanolprecipitation. Probe (10,000 cpm) and 11 microgram RNA were pooled,freeze dried, resuspended in 10 microgram hybridization buffer (40 mMpipes, pH 6.4; 1 mM EDTA, pH 8.0; 0.4 M NaCl; 80% (v/v) formamide),denatured for 10 min at 90° C. and annealed at 55° C. overnight. 235microliters ice-cold S1 nuclease buffer (0.25 M NaCl; 30 mM NaOAc; 1 mMZnSO₄ ; 200 micrograms single stranded calf thymus DNA) containing 2000units of S1-nuclease was added followed by an incubation at 37° C. for30 min. The reaction mixture was extracted once with phenol-chloroformand ethanol precipitated in the presence of 0.2M NaOAc and 10micrograms. yeast tRNA carrier. The resulting fragments were analyzed ona 6% (w/v) acrylamide, 7M urea DNA sequencing gel. For size standards,the Maxam Gilbert G and C sequencing reactions were performed on 50,000cpm of 5'- ³² P-labeled probe DNA. The results clearly show a protectedfragment and that the RNA start site is located at the C or T residue,position -86 or -87. A control indicates that in the absence of Z.ramigera RNA, the probe is completely degraded, demonstrating thepresence of the thiolase promoter regions approximately 10 bp (-96) and35 bp (-121) upstream. The 5'-untranslated region of the thiolase is 86bp long.

From the results of the induction experiments, it is clear that thethiolase gene can be expressed at high levels in a soluble,catalytically active form in E. coli. S1-nuclease studies map thetranscription start site for the thiolase gene in Z. ramigera atnucleotides -86/-87.

A possible explanation of the inhibitory effect of the thiolase promoterregion on tac-directed expression can be proposed based on theassumption that both promoters are recognized by RNA polymerase equallybut initiate transcription at very different rates. In this respect itis noted that the "-35" region of the thiolase promoter is very similarto the E. coli consensus sequence yet the thiolase "-10" region does notclosely resemble the TATAAT box, as shown in FIG. 3a and 3b. Studieshave demonstrated that although the "-35" region is recognized and bindsthe RNA polymerase, it is the "-10" region which determines the rate oftranscription initiation. In the case of pZT3, for example, the bindingof an RNA polymerase molecule to each promoter at the same time wouldresult in the rapid initiation of transcription from the tac promoterwhich would subsequently be impeded by the presence of the polymerasemolecule bound at the Zoogloea promoter. A consequence of thisexplanation is that the closer the two promoters are linked, the lesschance of polymerase binding to both at the same time and the lower theinhibition. Therefore, this represents one means for controlling rate ofexpression of the enzyme.

Identification of the Z. ramigera Reductase Gene

After identifying the promoter region of the thiolase gene and notingthe absence of any potential terminator sequences downstream from thethiolase TAG stop codon, the remaining 2 kb of Zoogloea DNA present inclone pUCDBK1 (FIG. 2) was sequenced and examined for the reductasegene. A series of expression plasmids (pZT1-pZT3) containing either theentire pUDCBK1 insert or fragments thereof were constructed in the E.coli tac promoter vector pKK223.3, as diagrammed in FIG. 5. Each plasmidhas the thiolase gene located in the correct orientation for expressionfrom the tac promoter. It is reasonable to expect the tac promoter todirect not only thiolase expression but the expression of any geneslocated in the 2.3 kb downstream in an operon-like type organization.Clone pZT1 was constructed by inserting the entire 3.8 kb EcoR1 Z.ramigera DNA insert from pUCDBK1 into the EcoR1 site of the vectorpKK223-3. Subsequently, pZT2 was derived from pZT1 in a straightforwardmanner by deleting the 850 bp Sma1 fragment. pZT3 is constructed asdescribed for the identification of the thiolase promoter. A series oftac promoter induction experiments were performed on each of therecombinant clones pZT1, pZT2 and pZT3. The vector pKK223-3 was used asa control.

E. coli cells containing each of the plasmids was grown and induced bythe addition of isopropyl-beta-D-galactopyranoside (IPTG) to a finalconcentration of 2 mM. After 15 h induction, 10 ml cultures wereharvested and lysed by sonication. The cell lysates from each clone werethen analyzed both by enzyme assay and on SDS-PAGE. No PHB polymeraseactivity was detected in any of these lysates. Each of the threerecombinant plasmids pZT1, pZT2 and pZT3 demonstrate substantial levelsof thiolase activity. In addition, the lysates from pZT1 and pZT2 havecomparably high levels of AcAc-CoA reductase activity using NADPH as thecofactor. No reductase activity is detected in any of the lysates whenNADH is used as a cofactor. The control, pKK223-3, has neither thiolasenor reductase activities. To confirm that the lysates from pZT1 and pZT2do in fact contain the correct reductase, these lysates were alsoassayed for oxidation of D(-)-hydroxybutyryl-CoA. In both cases, enzymeactivity was observed with NADP as electron acceptor.

Each of the lysates described above was also analyzed by SDS-PAGE. Theresult clearly show the presence of the thiolase protein at around42,000 daltons in protein lysates from pZT1, pZT2 and pZT3, which is notpresent in the control, pKK223-3. Lysates of pZT1 and pZT2 also have asmall, 25,000 dalton protein which is not present in the lysate pZT3 orthe control, assumed to be the AcAc-CoA reductase as the basis of therecent report by Fukui et al., (1987) of a subunit molecular weight forthe Z. ramigera AcAc-CoA reductase of 25,000. The results demonstratethat the AcAc-CoA reductase gene is located downstream from the thiolasegene. Furthermore, the entire structural gene for this enzyme must belocated between the 3'-end of the thiolase and the first Smal sitedownstream in pUCDBK1.

Identification of the Translation Start Site and Overexpression of theReductate Gene

The complete nucleotide sequence of the 2339 bp located 2.3 kbdownstream from the first Sal1 site in pUCDBK1 is shown in FIGS. 6a 6b.Computer analysis of the sequence data, using codon usage informationfrom the thiolase gene as a standard, identified three open readingframes. N-terminal protein sequence data was obtained from the 25,000dalton band present in induced lysates from pZT1 and pZT2 followingpreparative SDS-PAGE and electroelution. This data was used to confirmthe translation start site for the corresponding gene. The N-terminalfive amino acids determined experimentally match residues 2 through 6predicted from the DNA sequence of the first open reading frame.Translation of this reading frame predicts a polypeptide of 25,000molecular weight. The translation product of the first open readingframe starting at the ATG, nucleotide 37 and ending at the TGA stopcodon nucleotide is shown in FIGS. 6a and 6b. This is the predictedprimary amino acid sequence of the AcetoAcetyl-CoA reductase protein.

It is evident that the Acetoacetyl-CoA reductase gene in clones pZT1 andpZT2 can be expressed at reasonably high levels in E. coli. However, inboth of these cases, the expression of the reductase gene from the tacpromoter is not optimum due to the presence of the thiolase structuralgene and 5'-flanking sequence. Since there is a 5.9-fold inhibition oftac directed expression of thiolase promoter and both of these plasmidsexpress the thiolase gene at high levels which could present problems inpurification by affinity chromatography, a simpler Acetoacetyl-CoAreductase overproduction vector, pZR14, shown in FIG. 2, wasconstructed. pUCDBK1 DNA was digested to completion with Sal1 and Sma1and the Sal1 ends repaired using the Klenow fragment of DNA polymerase.Following the addition of EcoR1 linkers and digestion with EcoR1, thefragments were separated by agarose gel electrophoresis. The 1.05 kbfragment corresponding to the Acetoacetyl-CoA reductase structural geneplus 36 bp flanking the 5'-end and 266 bp flanking the 3' end waspurified and ligated into the EcoR1 site of pKK223-3. pZR14 was thenidentified as having the correct restriction map with the reductase genein the right orientation. Induction experiments were performed on pZT14as described for pZT1, pZT2 and pZT3. Acetoacetyl-CoA reductase wasexpressed.

Identification of the Thiolase and Reductase Genes in A. eutrophus

The methods used in isolating the first two PHB genes from Zoogloea wereapplied to the identification, isolation and characterization of genesfrom another PHB producing species, Alcaligenes eutrophus, using theZoogloea thiolase gene region as a hybridization probe to locatehomologous sequences.

Subsequent sequence analysis of a 2 kb Pst1 fragment of A. eutrophus DNAcloned into pUC8 (clone pAeT3), restriction mapped in FIG. 7 has clearlyidentified the corresponding thiolase gene region in the A. eutrophusH16 genome. The downstream sequences in pAeT3 are also homologous to theNADP-linked reductase gene region from the Zoogloea clone pUCDBK1. Thesequences of the Alcaligenes thiolase and reductase genes is shown inFIG. 8a and 8b.

Cloning of the thiolase and reductase genes from pAeT3 into pKK 223.3,as shown in FIG. 7, leads to expression of the corresponding enzymes.Comparisons of the Zoogloea and A. eutrophus thiolase protein sequencesestablish that the two proteins have a total of 68% identical residuesincluding the active site Cys-89.

Both the A. eutrophus and Zoogloea thiolase gene regions were used ashybridization probes to screen Nocardia DNA to identify thecorresponding Nocardia genes. Techniques for identifying the thiolase,reductase, and other polymerase genes from other species havinghomologous sequences in addition to those described above, are known tothose skilled in the art.

Identification of the Z. ramigera PHB polymerase gene

PHB polymerase from Z. ramigera utilizes D(-)-hydroxybutyryl-CoAmonomers, polymerizing them in oxoester linkages in atemplate-independent head to tail condensation to yield linear polymers.These polymers can contain up to 10,000 monomer units with a molecularweight in excess of 1×10⁶. The polymer rapidly becomes insoluble andaccumulates as discrete granules in the cell. Of fundamental interest isthe mechanism by which this unique enzyme polymerizes water solublemonomers, transporting them from the cytoplasm into a hydrophobic highlycrystalline granule.

Using a conjugal transfer system based on derivatives of the broad hostrange plasmid pRK290 described by Ditta et al., in Proc. Natl. Acad.Sci. USA 77, 7347-7351 (1980), transposon mutagenesis andcomplementation analysis can be performed in Z. ramigera. PHB negativemutants of Z. ramigera are isolated, characterized and thencomplemented. As described by Schlegel et al., Arch. Microbiol. 71,283-294 (1970), sudan-black staining is used for the detection of PHBnegative mutants. Complementation of the mutants is screened for bygrowing, harvesting, and lysing the cells to release PHB that can thenbe purified to determine its presence and molecular weight sizedistribution. Thiolase, reductase and PHB polymerase activities in thelysates are also assayed.

When the Sudan black screening technique was applied to Z. ramigera, atleast two Tn5 sudan black negative (PHB negative) mutants wereidentified and characterized. Construction of Tn5 mutant libraries isfacilitated by using an exopolysaccharide negative strain, Z. ramigeraS99 , described by Easson et al. (1987) submitted to the Journal ofBacteriology to overcome the problems of polysaccharide interferencewith the conjugation process and the screening procedure. Methods formutating and isolating Z. ramigera strains is described in ourco-pending application U.S. Ser. No. 035,604, filed Apr. 7, 1987, byEasson et al, entitled "Method to Control and Produce NovelBiopolymers".

A complete library of PHB mutants defective in each step of the pathwayhas been established. A similar Tn5 mutant library of A. eutophus H16has also been constructed. From the knowledge of the genomicorganization of the thiolase and the reductase genes, it is relativelystraightforward to screen PHB polymerarse mutants by Southernhybridization analysis using ³² P-labelled Tn5 as a probe to identifythe location of this gene. Complementation of the mutants is achievedusing the cosmid library described by Easson et al. (1987).

The polymerase complementing sequences are subcloned in astraightforward manner using techniques known to those skilled in theart on a smaller three to five kb restriction fragment for DNAsequencing. Computer analysis, utilizing the codon usage data fromthiolase and reductase as the Zoogloea standard can be used to locatethe protein coding regions. Sequences 5' to the potential translationstart signal, to be confirmed by protein sequence data, are analyzed forregulatory sequences and then the transcription initiation sitedetermined by S1-nuclease mapping.

Modification of Polymer Synthesis by Varying Levels of Enzyme Expression

After isolation and characterization of the polymer genes and geneproducts from a variety of organisms, as demonstrated for Z. ramigera,A. eutrophus, and N. salmonicolor, means for controlling the expressionof the gene products are established. Overproduction of the Zoogloeathiolase gene was demonstrated by the studies used to define thetranscription start site and promoter of the Z. ramigera. Overproductionenables the purification of the enzymes to homogeneity and providesreagent type quantities for analysis and comparison of substratespecificieties. In addition, the purified enzymes can be used tosynthesize stereospecific substrates for in vitro polymer synthesis.Further, once the transcriptional regulatory mechanism responsible forpolymer over-production is elucidated under a variety of environmentalconditions, in vitro systems for the enzymatic synthesis of knownpolymers, and novel polymers, can be developed to provide new materials.The new materials are then analyzed for chemical composition, molecularweight and rheological characteristics so that maximum utilization canbe made of the polymers.

An overproduction system for the Z. ramigera thiolase in E. coli wasconstructed using the synthetic tac promoter to produce a series ofthiolase expression plasmids, the optimum construct in induced E. colicells yielding about 20-30% of the total soluble cell protein asthiolase. This method yields thiolase in reagent type quantities, anaverage of 150 mg pure thiolase from 1 liter of culture. There areessentially two conditions where gene regulation in Z. ramigera and A.eutrophus may be expected to occur: when carbon starved cells undernutrient limiting conditions are subsequently presented with a carbonsource and when cells grown under nutrient limiting conditions haveaccumulated large amounts of PHB and the carbon source in the mediumbecomes depleted resulting in PHB degradation.

Transcriptional regulation of the polymer biosynthetic genes isdetermined as follows. Cultures grown under various conditions areharvested both for enzyme assays (thiolase, reductase and polymerase)and for RNA purification. RNA samples are analyzed in a series ofNorthern hybridization experiments using the cloned genes as probes.Useful RNA hybridization methodology includes glyoxylation of RNA(McMaster and Carmichael, Proc. Natl. Acad. Sci. USA 74, 4835 (1977));formaldehyde/agarose gel electrophoresis (Lehrach et al., Proc. Natl.Acad. Sci. USA 16, 4743 (1977)); transfer to nitrocellulose or nylonfilters (Thomas, Proc. Natl. Acad. Sci. USA 77, 5201 (1980)); andhybridization with DNA probes labelled with ³² P by nick translation(Rigby et al., J. Mol. Biol. 113, 237 (1977)). Probes are prepared fromclones pUCDBK1(Z. ramigera); and pAeT3 (A. eutrophus). One result ofthese studies is the establishment of the operon organization of thegenes and the length of the mRNA.

The levels of each of the biosynthetic enzymes are manipulated and theeffect this has on polymer synthesis monitored. It is necessary todetermine the rate-limiting enzyme(s) in the biosynthetic pathway sothat one can increase flux through the pathway by overproducing therate-limiting enzyme; the effect overproduction of each enzyme has onthe incorporation of different monomeric units, i.e., the ratio ofPHB:PHV in the copolymer produced by A. eutrophus when grown onbutyrate; and the result of expression of the genes from one species inother species, for example, the expression of Zoogloea genes in A.eutrophus , and vice versa, as well as other isolated and characterizedheterologous genes from other organisms, e.g., Nocardia and P.oleovarans in Zoogloea and A. eutrophus.

To accomplish overproduction of polymer biosynthetic genes in multiplehost organisms, one must use broad host range cloning vectors whichfunction in these bacteria. In one instance, enzyme overproduction viagene dosage is carried out. For example, the entire pUCDBK1 insertcontaining the promoter region can be cloned into the vector pSUP104(Simon et al., Molecular Genetics of the Bacteria-Plant Interaction, A.Pobler, ed. (Spring-Verlag, N.Y. 1983) and used to transform Z. ramigerI-16-M. The extent of overproduction of each enzyme is monitored byenzyme assays. A similar approach can be taken for any number of othergenes; for example, thiolase; thiolase and reductase; reductase;reductase and polymerase, etc. Secondly, genes can be placed under thetranscriptional control of high efficiency promoters, i.e., tac (Gill etal., J. Bact. 167, 611-615 (1986) and tol (Mermod et al., J. Bact. 167,447-454 (1986). In this case, the constructs are conjugated into mutantsdefective in the corresponding gene. The expression of the polymerbiosynthetic gene or genes of interest can then be tightly regulated, asdetermined using enzyme assays to monitor the level of overproduction.As each construct is tested, one can begin to monitor the effect onpolymer synthesis in a routine manner i.e., the rate and level ofsynthesis.

Modification of Polymer Synthesis by Altering Available Substrate orEnzyme Specificity

Factors which determine the molecular weight of the PHB produced bydifferent bacteria can be elucidated by analysing the molecular weightdistribution of the polymers produced by various bacteria. There islittle doubt that a number of PHB-producing microorganisms have theability to incorporate monomers other than D(-)-hydroxybutyrate into thepolymer chain. For the PHB-PHV copolymer produced by A. eutrophus , ithas been proposed that propionate is first converted to propionyl-CoAwhich then acts as a substrate for beta-ketothiolase. The high yields ofpure enzymes available from over-production systems is necessary todetermine the range of alternate substrates which each of the threePHB-biosynthetic enzymes can utilize and the types of new PHB-likepolymers that can be synthesized in an in vitro system where theavailable substrates can be strictly controlled.

Although the thiolase and reductase enzymes are an essential part of thebiosynthesis of PHB and PHB-like polymers, it is the PHB polymerasewhich ultimately defines the new types of polymer which can be made.This is facilitated by the development of an in vitro system using theenzyme to test a whole range of substrates, many which cannot enter thecell and therefore cannot be tested for incorporation into PHB in afermentation process.

Overproduction and purification of more than one reductase enzymeprovides a means for comparing the kinetics and specificity of theenzymes. The Zoogloea reductase has been reported to be NADP-specific,however, the A. eutrophus enzyme apparently will use either NAD or NADP.The stereospecificity of this enzyme may make it a useful reagent forthe synthesis of D-substrates for PHB synthetase studies. Among theacetoacetyl derivatives to be tested are the oxoester of CoA andoxopantetheine pivaloate (OPP) and the methylene analogs. The ketone butnot the oxoester of the methylene analogs is cleaved by Zoologeathiolase. Various longer chain alkyl derivatives where R does not equalH, and in particular the C₅ -C₈ linear 3-oxo thiolesters, oxoesters andmethylene ketones, may also be useful as substrates for the PHBsynthetase, given the existence of C₅ -C₈ -beta-hydroxyalkanoates in B.megaterium. We will also examine olefins, alcohols and epoxides.

In crude extracts of Z. ramigera, D-beta-hydroxybutyryl CoA, but notL-hydroxybutyryl CoA, is a substrate for PHB synthetase. It is expectedthat other D-hydroxyacyl CoA species will utilize alternate substratesor cosubstrates such as D-beta-hydroxyvaleryl CoA (HV-CoA). [2-3H]HB-CoAand beta [3-¹⁴ C]-HV-CoA, each readily preparable by enzymic or chemicalsynthesis, can be used as substrates and to monitor ³ H and ¹⁴ C contentand ratios in polymers precipitated or separated by gel filtration. Itis predicted that block copolymer regions, e.g., (HB)₅₀₀ (HV)₁₀₀₀(HB)₅₀₀, can be constructed by careful control of substrate ratios, andleaving groups in elongation phase, e.g., HB-oxo-CoA and HV-S-CoAmonomers.

Additional alternate substrates can be tested including branched chainbeta-hydroxyacyl CoAs. Testing cannot be done in whole cells since suchcompounds are not normally transported. Alternate substrates will betested for inhibition of normal [¹⁴ C]-PHB formation first byincorporation of soluble [¹⁴ C]-HBCoA into insoluble polymer, then ascopolymerization cosubstrates and finally for homopolymerization.Alternate substrates will be assayed for K_(m), V_(max) relative toHB-CoA and for polymer size determined by calibrated gel filtrationstudies.

FIG. 9 summarizes some of the monomeric acyl CoA analogs ofD-3-hydroxybutyryl-CoA to be tested as substrates for PHB polymerase andalso diagrams a proposed repeating unit that would arise fromhomopolymeric polyester formation or of a block region in a blockcopolymer from enzymatic co-incubation with HB-CoA to yield PHBformation itself. Entry 2 is the isomeric 2-hydroxybutyryl CoA and wouldyield a diagnostic ethyl signal by NMR and a 1,2-ester link rather thana 1,3 link. This would alter polymer properties even at a low molepercent incorporation. Entry 3 is the 4-hydroxybutyryl CoA isomer andwould yield a longer 1,4-ester link, possibly more crystalline. Entry 4is known from in vivo work to be a substrate for copolymerization withentry 1. It may actually serve as a substrate for homopolyesterproduction with either Zoogloea or Alcaligenes pure PHB synthase. Entry5 is the corresponding 2--OH C₅ substrate and yields both 1,2-1inkageand propyl branching, detectable by proton and ¹³ C NMR.

Entries 6 and 7 introduce C₄ and C₅ vinyl branched units in 1,2 and1,3-ester linkages (detectable by IR) and entry 8 introduces anepoxy-functionalized polyester. These three products can be subjected toradical-initiated cross-linking to covalently attach adjacent polyesterchains by cross-linking. Lastly, entries 9 and 10 probe stericrequirements at C₃ and C₂ for PHB synthase. They yield homopolymers orregions of copolymer even less crystalline than poly HB/HV, which is abetter performing thermoplastic material than PHB.

Substrates listed in FIG. 9 are representative acyl thiolestercandidates for PHB synthetases. They are straightforward to prepare andeasily assayed. Promising results can be followed by ¹⁴ C or ¹³C-enrichment for more quantitative analysis of mole fractionincorporated into polyester product. It is extremely likely that anumber of these analogs will be polymerized. The initial rates(assayable by ¹⁴ C-incorporation or by dithiobisnitrobenzoate-titratableCoASH release), size of product (gel filtration, radioactivity analysis)and material properties can then be evaluated.

Physical and Chemical Analysis of PHB and PHB-like Biopolymers

For chemical and physical properties analysis, the materials are firstpurified from fermentation processes or from enzymatic reactions. In thecase of whole cells, the biopolymer is extracted by the sodiumhypochlorite method. Polymers synthesized in vitro are extracted withchloroform.

Purification of PHB Granules

Concurrent with the molecular studies, large quantities of PHB granulesfrom Z. ramigera are purified according to the method of Fukui et al,Arch. Microbiol. 110, 149-156 (1976). This procedure results in yieldsof over 10 gram of PHB per liter cells. Protein containing PHBpolymerase activity, which accounts for between 1 and 2% of thegranules, is extracted by mild alkali treatment as described by Griebeland Merrick, J. Bacteriol. 108, 782-789 (1971). Analysis of the proteinby SDS-polyacrylamide gel electrophoresis is of use in identifying thesize and number of polypeptides associated with the PHB granules.Preparative SDS-PAGE and electroelution of protein bands is used toprovide material for NH₂ -terminal sequence analysis, thereby permittingthe design of oligonucleotides and the preparation of antibodies forimmunological screening, as described for the thiolase.

In an example of the purification of overproduced thiolase, plasmidcontaining E. coli JM105 cells were grown in 2×TY medium containing 50microgram/ml ampicillin. One liter of cells was grown to an A₅₉₅ of0.6-0.7 and induced by the addition of IPTG to a final concentration of2 mM. Inductions were performed for 16 h and the cells harvested bycentrifugation. All remaining procedures were carried out at 4° C. withpre-cooled solutions. Cell pellets were resuspended in 30 ml of lysisbuffer (20 mM Tris HC1, pH 8.0; 5 mM EDTA; 5 mMbeta-mercaptoethanol;0.02 mM phenylmethylsulfonyl fluoride; 5% (v/v) glycerol) and lysed bysonication. Cell debris was subsequently removed by centrifugation.

DEAE CL6B Sepharose (Pharmacia Fine Chemicals, Piscataway, N.J.) wasactivated as per manufacturers instructions and pre-equilibrated withlysis buffer. Two methods were used for this step. A batch procedure wascarried out by mixing one-half of the lysate supernatant with 25 ml ofpacked DEAE CL6B Sepharose resin for 10 min to bind the protein. Afterbinding, the resin was washed with 2×25 ml lysis buffer and 2×25 mllysis buffer containing 75 mM KCl. Thiolase containing protein wassubsequently eluted with 3×25 ml lysis buffer containing 0.2 M KCl, withincubations for 30 min, 1 h and 12 h. Alternatively, a DEAE CL6B column(2.5 cm×25 cm) was set up and eluted with a linear 0 to 1M KCl gradient(300 ml of each in lysis buffer).

Reactive Red 120-agarose type 3000-CL (Sigma, St. Louis, Mo.) wasactivated with 1 column volume (2.5 cm×35 cm) of 7M urea in 0.5M NaOHand equilibrated with lysis buffer containing 0.2M KCl. Eluant from theDEAE step was loaded directly onto the Red gel column without priortreatment and washed with 2 column volumes of 0.2M KCl in lysis buffer.Protein was eluted with a linear 0.2-2M KCl (30 ml each) gradient inlysis buffer. Fractions containing thiolase were pooled and dialyzedagainst 4 liters of 10 mM Tris HC1, pH 7.5; 5 mM beta-mercaptoethanol; 1mM EDTA. Purified protein was stored at 4° C. in dialysis buffercontaining 50% w/v) glycerol.

Purification of the native PHB polymerase from granules has not beenvery successful. In contrast, the soluble enzyme can be purified 50-foldto a specific activity of 1.1 mole substrate polymerized/min/mg via DEAESepharose and hydroxyapatite chromatography. A strategy ofchromatography on DEAE followed by affinity chromatography on variousligand-substituted Sepharoses, such as oligohydroxybutyryl Sepharose,Octyl Sepharose 4B, and butyl toyopease (Toyo Soda Co.) can be employedto purify the overproduced enzyme, using methods known to those skilledin the art.

The following assays and parameters must be determined to fullycharacterize the purified polymers: chemical analysis; molecular weightdistribution: hydrodoynamic volume; viscosity; chain conformations;chain interactions; and time functions.

Subunit composition is assayed by a method involving treatment of thepolyesters with ethanolic HCl, followed by GC analysis of thehydroxyacyl ethyl esters. Where appropriate, fractions are monitored forradioactivity. The detection limit is 100 femtamoles (10⁻¹² moles) androutine analysis has been reported on 100 nanograms of polymer (Findlayand White, Appl. Environ. Micro. 45, 71-78, 1983). PHB and relatedpolymers have relatively simple repeating units compared with the moreusual biopolymers such as proteins or polysaccharides. The repeatingunits of 4 or 5 carbon atoms can be established by ¹ H and ¹³ C NMR, byanalyzing the number and relative positions. of NMR signals, incombination with the proton-proton and carbon-proton coupling patterns.The repeating units, 4 or 5 carbons, are indicated by the ¹³ C NMRspectra. Homopolymer structure can be confirmed with NMR analysis. Forheteropolymers, the relative content of the different monomers can beevaluated by their relative intensitites in the NMR spectrum. Forexample, the relative areas of the two methyl groups at σ=0.85 andσ=1.21 can be used to determine the relative content of the two monomersin bacterial poly beta-hydroxybutyrate co-beta-hydroxypentanoate (Caponet al., Phyto Chemistry 22, 1181-1184 (1983); Bloemberge et al., Am.Chem. Soc. Div. Polym. Chem. 27, 252 (1986)). Monomer sequence can alsobe determined in some cases by NMR analysis (Grasdalen et al.,Carbohydrate Res. 68, 23-31 (1979); Iida et al., Macromolcules 11,490-493 (1978)).

The molecular weight distribution of PHB polymers is determined bycalibrated gel filtration studies (Barham et al., J. Materials Sci. 19,2781-2794 (1984); Suzuki et al., Appl. Microbiol. Biotechnol. 23,322-329 (1986)). The gel permeation mobilities have been related toabsolute molecular weights using molecular weight-dependent intrinsicviscosity by Mark-Houwink-Sakurada parameters. Columns aremicrostyragel, run in CHCl₃ at 30° C., analyzed with I.R. detector andcalibrated with polystyrene (Barham et al., (1984)).

The hydrodynamic volume of individual molecules of PHB can be evaluatedfrom the intrinsic viscosity determined from dilute solutions of PHB.The viscosity of suspensions of particles are described in terms of thedifference between the macroscopic viscosity n' of the suspension andthe viscosity n of the pure solvent. Specific viscosity, n_(sp)≡(n-n)/n. The specific viscosity, in the limit of infinite dilution,should be proportional to the number of suspended particles per unitvolume. In a macromolecular solution, n_(sp) is proportional to theconcentration, C, in grams per cubic centimeter. Thus n_(sp) /C, reducedviscosity, should be independent of concentration, as it is the limit ofzero concentration. This limiting value of n_(sp) /C gives intrinsicviscosity [n], ##EQU1##

The intrinsic viscosity can be determined by measuring (n'-n)/nC atvarious concentration and extrapolating to C=0. Alternatively, theintrinsic viscosity can be obtained by measuring (1/C)ln(n'/n) andextrapolating to zero concentration. For, ln(n'/n)=[1+(n'/n)/n]. As thelimit of zero concentration is approached, (n'/n)/n becomes very small,and the logarithm may be replaced by (n'-n)/n; i.e., ##EQU2##

The viscosity of dilute solutions of PHB are measured using aFenske-Cannon type capillary viscometer. The time, t, for a given volumeof liquid, V, as defined by the two etched lines in the Viscometer, toflow through the capillary of length, L, and radius, r, is measured. Ifthe density of the liquid, p, is known, the viscosity can be calculatedfrom the Poiseuille equation: ##EQU3## where n is the absolute viscosityand P, the applied pressure differential is defined by P=hgp, where h isthe average height of the liquid in the tube and g is the accelerationdue to gravity. In practice, the flow time for a liquid of knownviscosity is measured and a viscometer constant, A, is determined suchthat the kinematic viscosity (Y=n/p) and A are defined by the equations:##EQU4##

Chain conformation of PHB and related polymers are estimated by themolecular weight dependency of intrinsic viscosity as indicated by theMark-Houwink constant, a, as follows:

    [n]=K(MW).sup.a

The Mark-Houwink constant indicates the progression of polymer backboneas the molecular weight increases and therefore can describe the overallchain conformation. A Mark-Houwink constant close to zero indicatesrigid spherical shape conformation, between 0.5 and 0.7 indicatesequivalent sphere random coil, 0.7 and 1.0 indicates free drainingrandom coil and 1.2 or above indicates rigid-rod conformation. Suchinformation is critical for applications and fabrication procedures forsuch molecules. Polymer produced under various conditions (i.e.,fermentation time) or polymer subjected to sonication or enzymaticdegradation will be used to determine the intrinsic viscosity at a widerange of molecular weights. The Mark-Houwink constants for PHB andrelated polymers are also a function of the test solvent and thereforeintrinsic viscosity measurements must be made over several molecularweight values.

Polymer-polymer interactions are evaluated by steady shear viscositydetermined for various concentrations of a given biopolymer. The steadyshear viscosity-concentration with C[n]curve, as high as the solubilitypermits, is constructed to evaluate intermolecular chain interactions.The ratio of the concentration at which viscosity concentration deviatesfrom linearity and the reciprocal of the intrinsic viscosity will beused to represent interchain interactions.

Polymer-polymer chain interactions are evaluated by the viscoeleasticbehavior. The storage and loss modules can be determined in thefrequency range of 10⁻³ to 10 Hz using a Bohlin Rheometer System (Lund,Sweden). Assuming that the rubber elasticity holds in the rubberyregion, the length of chain segment between the crosslinks, N_(c), isestimated according to Florey, Principles of Polymer Chemistry, Chapter11, pages 432-494 (Cornell Univ. Press, Ithaca, N.Y., (1953) using theelastic modulus (G') from rubbery regions where G' is independent offrequency. The frequency at which elastic modulus (G') equals lossmodulus (G') for various concentration of polymers should be evaluatedalso to indicate degree of chain interations. ##EQU5##

The viscosity of polymers as a function of shear ratio (0.01 to 300sec⁻¹) is determined at 25° C. using a Bohlin Rheometer System (Lund,Sweden). The lower Newtonian, pseudoplastic and higher Newtonian regionshould be determined. Characteristic time is estimated from thereciprocal of the shear rate at which lower Newtonian region ends. Thehysteresis effect is determined by subjecting polymer solutions torepeated shear cycles. The relaxation time is determined directly usingthe Bohlin Rheometer.

Modifications and variations of the present invention, a method formaking polyhydroxybutyrate and polyhydroxybutyrate-like polymers havingcarbon-carbon backbones using recombinant engineering according to theforegoing detailed description, and the resulting polymers, will beobvious to those skilled in the art. Such variations and modificationsare intended to come within the scope of the appended claims.

We claim:
 1. An isolated DNA sequence hybridizing to a gene encodingbeta-ketothiolase when incubated for a period of approximately 16 to 18hours at a temperature of 65° C. in a mixture of a 5x solution of 0.15MNaCl, 0.15M sodium citrate, 20 mM sodium phosphate, 5x Derthardt'ssolution, 0.1% (w/v) SDS, 10 mM EDTA, and 100 μg/ml sonicated denaturedsalmon DNA.
 2. The DNA sequence of claim 1 which hybridizes to the geneshown in FIG. 3 or 8 when incubated for a period of approximately 16 to18 hours at a temperature of 65° C. in a mixture of a 5x solution of0.15 M NaCl, 0.15M sodium citrate, 20 mM sodium phosphate, 5x Denhardt'ssolution, 0.1% (w/v) SDS, 10 mM EDTA, and 100 μg/ml sonicated denaturedsalmon DNA.
 3. The DNA sequence of claim 2 having the sequence shown inFIG. 3 or conservative substitutions thereof.
 4. The DNA sequence ofclaim 2 having the sequence shown in FIG. 8 or conservativesubstitutions thereof.
 5. An organism genetically engineered to expressthe gene of claim 1 encoding beta-ketothiolase which occurs naturallyand is expressed in a bacteria selected from the group consisting ofZoogloea, Bacillus, Nocardia, Clostridium, Halobacterium, Pseudomonas,and Alcaligenes.